Abstract

Recent advances in CRISPR/Cas-based genome editing, including three-component CRISPR, the use of long single-strand donors, enhanced microhomology-mediated end-joining (MMEJ), and pharmacological approaches have greatly improved knock-in (KI) efficiency. In our search for factors to further improve KI efficiency for broad therapeutic use and efficient generation of primate disease models, we found that the strand exchange protein RAD51 can significantly increase homozygous KI efficiency using CRISPR/Cas9 in mouse embryos through an interhomolog repair (IHR) mechanism. Using a variety of approaches, we demonstrate robust enhancement of zygotic IHR by RAD51 and show that this can be leveraged both for generating homozygous KI animals from wildtype zygotes with exogenous donors and for converting heterozygous alleles into homozygous alleles without exogenous templates. Thus, our study provides both conclusive evidence supporting the existence of zygotic IHR mechanisms and a new method to significantly improve IHR efficiency for potential therapeutic use.

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